Fig. 1: CALM shRNA severely impairs the growth of hematopoietic cells with MT-RTKs.

a CALM was knocked down in Ba/F3-FLT3 WT, Ba/F3-FLT3 ITD, Ba/F3-KIT WT, and Ba/F3-KIT D814V by shRNA specific to CALM (CALM shRNA) or scrambled (SCR) shRNA as a control. These clones were cultured under various conditions to assess the influences of CALM KD on IL-3-dependent growth (left panel), FL- and SCF-dependent growth (center panel), and FLT3 ITD- and KIT D814V-dependent growth (right panel). The growth of these cells was assessed at the indicated points. Data shown are the mean ± SEM from three independent experiments. Two-sided unpaired Student’s t test, *p = 0.0018, **p = 0.0018. b CALM was knocked down with SCR shRNA as a control in AML cell lines, MV4-11, HMC-1, and HL-60, with a doxycycline (DOX) inducible system. The growth of these cells was monitored until 72 h after the start of DOX treatment. Data shown are the mean ± SEM from three independent experiments. Two-sided unpaired Student’s t test, *p = 0.0013, **p = 0.0003. c Tyrosine-phosphorylated status of FLT3, KIT and their downstream STAT5, and Akt were investigated by immunoprecipitation and/or immunoblot analyses using the indicated cells. Representative images of two independent experiments are shown. Densitometry analysis was carried out by Image Quant TL, and data is from two independent experiments.